All cells are delimited by a plasma membrane, which forms a permeability barrier between the intracellular machinery and the extracellular environment. We are interested in those components of the cell membrane, especially transmembrane proteins/receptors, that initiate the response of cells to external stimuli. The response of cells to the polypeptide hormone insulin commences upon the binding of the ligand to the insulin receptor (IR), a transmembrane glycoprotein. We would ultimately like to be able to relate the features of the primary structure of the IR, revealed from the deduced amino acid sequence, to the details of insulin receptor function. Given the large size (1355 amino acids) and membrane-associated nature of the IR molecule, it is experimentally advantageous to deal with the two major functional domains of the IR (the extracellular, ligand-binding domain, and the cytoplasmic, protein-tyrosine kinase (PTK) domain) as two discrete soluble molecules. We are therefore now exploiting our ability to express and study the two domains independently, as a secreted extracellular and a cytoplasmic PTK domain, respectively. The objectives of the present proposal are to overexpress (via the cDNAs) soluble and fully functional IR PTK domains (wild-type and a series of judiciously chosen, site- directed point mutations) in a heterologous expression system (the Baculovirus insect cell system) to study the functional properties of this class of enzyme and to attempt to crystallize these enzymes for future elucidation of their three-dimensional structure. A detailed investigation of the structure/function of the IR PTK domain is expected to contribute significantly to our understanding of the mechanism(s) of action not only of the IR, but also of a related family of ligand-activated transmembrane receptors and transforming proteins that harbor PTK domains, and are implicated in the control of cell growth and proliferation.